The widespread use of antimicrobial chemotherapeutics has had the inevitable consequence of the emergence of antibiotic-resistant pathogens, which has continualy prompted further development and design of new drugs to combat such organisms. Today, more than 70% of the bacteria associated with nosocomial infections in the United States are resistant to one or more of the drugs previously used to treat them. Drug resistance in bacteria—which is not limited to the U.S. but extends throughout the world—includes, for example, the worldwide emergence of Haemophilus and gonococci that are resistant to β-lactam antibiotics (e.g., penicillin), methicillin-resistant Staphylococcus aureus, multiple-drug resistant S. aureus with high-level resistance to vancomycin, various isolated strains of Pseudomonas and Enterobacter that are resistant to all available antibiotics, and multiple-drug resistant strains of Mycobacterium tuberculosis. 
While society increasingly has recognized the negative consequences of the misuse of antibiotics, overuse and overprescribing of antibiotics continue to be widespread througout the world, driven by diagnostic uncertainties, demands by patients, and physicians' lack of time to effectively evaluate patients. Although reducing inappropriate antibiotic use is thought to be the best way to curb resistance, physicians must generally be more selective and prudent in their use and prescribing of antibiotics so that the gains in the battle against infectious diseases over the past century are not lost. The rampant spread of antibiotic resistances mandates a more responsible and sensible approach to antibiotic use.
The β-lactam antibiotics (e.g., β-lactam ring-containing antibiotics, such as, penicillins, cephalosporins, or carbapenems, which inhibit bacterial cell wall synthesis) are a particularly prevalent and important class of antibiotics that are widely prescribed for a large variety of gram-negative and gram-positive infections. Consequently, widespread resistance has emerged. One particularly important mechanism of microbial resistance to the β-lactam antibiotics stems generally from the production of enzymes known as the β-lactamases or cephalosporinases, which enzymatically cleave β-lactam antibiotics thereby causing their inactivation. This type of resistance can be encoded on the chromosome or on extra-chromosomal elements (e.g. bacterial plasmids), which can be transferred horizontally to other bacteria. Other modes of resistance to β-lactam antibiotics also include acquisition of penicillin-binding proteins and decreased entry and/or active efflux of drugs through membrane efflux pump systems.
Clinical detection of β-lactamases represents a key step in the management of antibiotic therapy of bacterial infections. In particular, the amount of beta-lactamase activity and the substrate specificity of that activity are important considerations in determining the appropriate antibiotic therapy for patients suffering from drug resistant bacterial infections.
β-lactam susceptibility and resistance can be detected and/or measured in a variety of ways. For example, Kirby-Bauer antibiotic testing uses antibiotic-impregnated discs to test whether particular bacteria are susceptible to specific antibiotics. A known quantity of bacteria is grown on agar plates in the presence of thin wafers containing relevant antibiotics, such as penicillin or ampicillin. If the bacteria are susceptible to the antibiotics, a zone of inhibition forms around the diffusion zone of the wafers. The size of the zone is correlated to the minimum inhibitory concentratin (“MIC”) of antibiotic for that bacteria. In this way, health care providers are able to choose appropriate antibiotics to combat a particular infection.
In addition, agar dilution methods and broth microdilution methods can be used. Many of these labor-intensive and time-consuming methods often fail to detect drug resistance in certain gram-negative bacteria and all require a pregrowth step in which the strain is grown in broth or on a plate under conditions in which the organism is exposed only to the inducing antibiotic. This step is followed by a challenge in the presence of an indicator antibiotic or direct assay of enzymatic activity. These approaches require pure culture inoculation and growth, and involve up to 24 hours of incubation.
Chromogenic substrates have also been used, which, when cleaved by bacterial beta-lactamases, produce a colorimetric change that can be detected or measured. Examples of such substrates include, for example, nitrocefin and centa, which are known in the art. Nitrocefin is sold in the form of impregnated paper discs, which, when placed in the vicinity of a bacterial culture producing β-lactamase, results in the development of a pink color. Although this method provides a rapid qualitative detection (i.e., yes/no), it does not provide any information regarding the relative amount of enzymatic activity or any insight into the type of beta-lactamase activity, and, thus, cannot be used alone to determine the appropriate course of therapy in a clinical setting.
As can be seen, currently available methods for detecting and evaluating beta-lactamase activity and resistance to beta-lactam antibiotics suffer a number of drawbacks. In particular, such methods, while providing qualitative information (yes/no) about drug resistance, are time-consuming and do not easily facilitate the quantitative measurement of enzyme activity or determination of enzyme type or substrate characteristics—which constitute highly valuable information for determining an appropriate strategy for antimicrobial therapy.
Accordingly, new methods and compositions for detecting and evaluating beta-lactamase activity, which are more sensitive, rapid and easier to perform, and which reliably and expediently provide both qualitative and quantitative information as to the activity and/or substrate specificity of a beta-lactamase, would represent an advance in the art.